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K Takeshige

Publications and source records attributed to K Takeshige.

At least 55 records · Page 3Linked to original sources
PubMed1994

Role of Src homology 3 domains in assembly and activation of the phagocyte NADPH oxidase.

The phagocyte NADPH oxidase, dormant in resting cells, is activated during phagocytosis to produce superoxide, a precursor of microbicidal oxidants. The activated oxidase is a complex of membrane-integrated cytochrome b558, composed of 91-kDa (gp91phox) and 22-kDa (p22phox) subunits, and two cytosolic factors (p47phox and p67phox), each containing two Src homology 3 (SH3) domains. Here we show that the region of the tandem SH3 domains of p47phox (p47-SH3) expressed as a glutathione S-transferase fusion protein inhibits the superoxide production in a cell-free system, indicating involvement of the domains in the activation. Furthermore, we find that arachidonic acid and sodium dodecyl sulfate, activators of the oxidase in vitro, cause exposure of p47-SH3, which has probably been masked by the C-terminal region of this protein in a resting state. The unmasking of p47-SH3 appears to play a crucial role in the assembly of the oxidase components, because p47-SH3 binds to both p22phox and p67phox but fails to interact with a mutant p22phox carrying a Pro-156-->Gln substitution in a proline-rich region, which has been found in a patient with chronic granulomatous disease. Based on the observations, we propose a signal-transducing mechanism whereby normally inaccessible SH3 domains become exposed upon activation to interact with their target proteins.

Amino Acid Sequence↗
PubMed1994

Prognosis after hepatic resection in patients with hepatocellular carcinoma, estimated on the basis of the morphometric indices.

To determine whether the morphometric indices of hepatocellular carcinoma (HCC) correlated with the prognoses, the microscopic morphometric values for 84 HCC cases treated by hepatic resection were studied using an image analyzer in relation to the survival rate and the gross classification. The mean survival time (MST) was 58 months in cases with a nucleocytoplasmic area ratio (N/C) of less than 0.28; this was significantly longer than the 38-month MST in cases with an N/C of more than 0.28 (P < 0.05). In stage III disease, the MST for cases with an N/C of less than 0.28 was 63 months, which was significantly longer than the MST of 13 months for cases with an N/C of more than 0.28. After relatively noncurative hepatic resection, the MST for cases with an N/C of less than 0.28 was 49 months, and this was significantly longer than the MST of 8 months for cases with an N/C of more than 0.28. The MST was 71 months for cases with a coefficient of variance of the nuclear form factor (NCV) of less than 5.5%, which was significantly longer than the MST of 33 months for cases with an NCV of more than 5.5% (P < 0.05). In stage III disease, the MST was 69 months for cases with an NCV of less than 5.5%, and this was significantly longer than the MST of 29 months for cases with an NCV of more than 5.5% (P < 0.05). In cases with an N/C of less than 0.28, 18% had vascular invasion and 38% had intrahepatic metastases, whereas in those with an N/C of more than 0.28, 62% had vascular invasion and 67% had intrahepatic metastases (P < 0.01, P < 0.05). Based on the results of these morphometric studies on HCC cases treated by hepatic resection, N/C and NCV may be useful as prognostic factors.

Carcinoma, Hepatocellular↗
PubMed1994

Ultrastructural analysis of the autophagic process in yeast: detection of autophagosomes and their characterization.

Under nutrient-deficient conditions, the yeast S. cerevisiae sequesters its own cytoplasmic components into vacuoles in the form of "autophagic bodies" (Takeshige, K., M. Baba, S. Tsuboi, T. Noda, and Y. Ohsumi. 1992. J. Cell Biol. 119:301-311). Immunoelectron microscopy showed that two cytosolic marker enzymes, alcohol dehydrogenase and phosphoglycerate kinase, are present in the autophagic bodies at the same densities as in the cytosol, but are not present in vacuolar sap, suggesting that cytosolic enzymes are also taken up into the autophagic bodies. To understand this process, we performed morphological analyses by transmission and immunological electron microscopies using a freeze-substitution fixation method. Spherical structures completely enclosed in a double membrane were found near the vacuoles of protease-deficient mutant cells when the cells were shifted to nutrient-starvation media. Their size, membrane thickness, and contents of double membrane-structures corresponded well with those of autophagic bodies. Sometimes these double membrane structures were found to be in contact with the vacuolar membrane. Furthermore their outer membrane was occasionally seen to be continuous with the vacuolar membrane. Histochemical staining of carbohydrate strongly suggested that the structures with double membranes fused with the vacuoles. These results indicated that these structures are precursors of autophagic bodies, "autophagosomes" in yeast. All the data obtained suggested that the autophagic process in yeast is essentially similar to that of the lysosomal system in mammalian cells.

Alcohol Dehydrogenase↗
PubMed1994

Phosphatidic acid induces the release of beta-glucuronidase but not lactoferrin from electropermeabilized human neutrophils.

We studied the degranulation reaction of electropermeabilized human neutrophils induced by 1,2-didecanoyl-3-sn-phosphatidic acid (PA10). PA10 dose-dependently induced the release of beta-glucuronidase, an enzyme of azurophil granules, but did not induce the release of lactoferrin, a protein of specific granules. The enzyme release by PA10 absolutely required Ca2+, ATP, and Mg2+ and the concentrations for the half-maximal response were 2.5 microM, 60 microM, and 0.25 mM, respectively. Although Ca2+ alone at concentrations higher than 10 microM induced the release of both beta-glucuronidase and lactoferrin, the extents of the release were far less than that of the beta-glucuronidase release by PA10. Phorbol myristate acetate (PMA) and 1-oleoyl-2-acetyl-sn-glycerol induced the release of lactoferrin alone at concentrations of Ca2+ below 0.5 microM while they induced the release of both beta-glucuronidase and lactoferrin at higher Ca2+ concentrations, indicating that the degranulation induced by PA10 is not mediated by diacylglycerol which might be formed from PA. The degranulation reactions induced by PA10 and PMA were dose-dependently inhibited by staurosporine and calphostin C, protein kinase C inhibitors, although no direct activation of protein kinase C by PA10 was observed. The extent of the beta-glucuronidase release by PA10 was not enhanced by the addition of PMA. Propranolol, which inhibits protein kinase C as well as phosphatidic acid phosphohydrolase, strongly inhibited the degranulation reactions induced by PA10 and PMA. Ethanol, a metabolic modulator of phospholipase D, and cyclic AMP did not affect the degranulation reactions by PMA and PA10.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium↗
PubMed1994

Adenine nucleotide metabolism in relation to graft viability in rat small-bowel transplantation.

The relation between the level of cellular adenine nucleotides and graft viability after cold preservation and warm ischemia was studied by using a rat small-bowel transplantation model. The rat jejunum was preserved in lactated Ringer's solution at 4 degrees C for 120 min, and was then transplanted syngeneically under warm ischemia after between 60 and 180 min. The recovery levels of ATP and the energy charge at 30 min after reperfusion in the viable graft were significantly higher than those in the nonviable graft. None of the grafts were viable when the recovery level of ATP at 30 min after reperfusion was less than 2.8 mumol/g dry weight. These results suggest that the recovery level of ATP at 30 min after reperfusion is a reliable parameter for graft viability in small-bowel transplantation.

Adenine Nucleotides↗
PubMed1994

[Superoxide formation and lipid peroxidation by the mitochondrial electron-transfer chain].

Isolated mitochondria supplemented with succinate or NAD(+)-linked substrates generate hydrogen peroxide (H2O2) in State 4 and the generation is enhanced by antimycin A, an inhibitor of the respiratory chain. Superoxide is a stoichiometric precursor of mitochondrial H2O2 because the ratio of O2-/H2O2 generation rates is close to 2.0 and is generated by an autoxidizable component in the NADH dehydrogenase and the ubiquinone-cytochrome b site. Lipid peroxidation is a free radical-mediated degradation of polyunsaturated fatty acids. Lipid-peroxidation reactions by bovine submitochondrial particles are supported by NADH or NADPH in the presence of ADP-Fe3+ chelate. Electrons from NADH are supplied to the reactions from a component between the substrate site and the rotenone-sensitive site of the NADH dehydrogenase. The peroxidation is dependent on the rate of electron input into the respiratory chain and on the concentration of reduced ubiquinone. Alteration of inner-membrane components and damage to electron-transfer activities of submitochondrial particles are induced by lipid peroxidation. 1-Melhyl-4-phenylpyridinium (MPP+), a metabolite of a parkinsonism-inducing drug, induces NADH-dependent superoxide formation and enhances NADH-dependent lipid peroxidation in submitochondrial particles, indicating that the oxidative stress induced by MPP+ may potentiate its toxicity in dopamine neurons.

Electron Transport↗
PubMed1993

Decrease in the phosphotyrosine phosphatase activity in the plasma membrane of human neutrophils on stimulation by phorbol 12-myristate 13-acetate.

Phorbol 12-myristate 13-acetate (PMA) induced a decrease in the phosphotyrosine phosphatase (PTPase) activity in human neurophils. The decrease in the activity induced by PMA was blocked by the treatment of the cells with staurosporine, indicating that protein kinase C is involved in the decrease. The PTPase activity was localized in the plasma membrane. The activity in the membrane with the optimum pH at 5.5 had a Km value for phosphotyrosine of 2.2 mM and Vmax of 2.0 mumol/min per mg of protein. No activity was observed against phosphoserine and phosphothreonine. Vanadate, molybdate, zinc and a sulfhydryl reagent, p-chloromercuribenzenesulphonic acid, inhibited the PTPase. The PMA-induced decrease in activity was almost completely recovered by treatment of the plasma membrane with Triton X-100 at low concentrations which did not solubilize the activity from the membrane. When the plasma membrane was treated with trypsin, the PTPase of the membrane from PMA-treated cells was mostly protected from the proteinase attack while that from the resting cells was not protected. Pretreatment of the plasma membrane with Triton X-100 enabled trypsin to gain access to all the PTPase in the membrane from both PMA-treated and resting cells. The PMA treatment affected neither subcellular localization of the PTPase nor the orientation of the plasma membrane vesicles. These findings suggest that conformational changes of the enzyme induced by PMA result in the decrease in PTPase activity.

Alkaloids↗
PubMed1993

Phosphatidic acid induces the respiratory burst of electropermeabilized human neutrophils by acting on a downstream step of protein kinase C.

Phosphatidic acid (PA) dose-dependently induced superoxide (O2-) production of electropermeabilized human neutrophils but not of intact neutrophils, indicating that PA induces the activation of NADPH oxidase by acting on an intracellular target. The O2- production by PA was not inhibited by protein kinase C (PKC) inhibitors, such as staurosporine and calphostin C, and an inhibitor of PA phosphohydrolase, propranolol. These observations suggest that the activation of the oxidase by PA is independent of the activity of PKC and may dominate the activation by diacylglycerol which is formed from PA via the action of PA phosphohydrolase. Furthermore, the production by PA, as well as that by phorbol myristate acetate, was inhibited by cyclic AMP and GDP beta S. Therefore, PA seems to act at a site downstream of PKC.

Cell Membrane Permeability↗
PubMed1993

Characterization of the GTP-dependent activation of the superoxide-producing NADPH oxidase in a cell-free system of pig neutrophils.

We characterized the cell-free activating system of the superoxide (O2-)-producing NADPH oxidase of pig neutrophils. Activation of the oxidase required both the membrane and cytosolic fractions in the presence of sodium dodecyl sulfate. Chromatography on 2',5'-ADP-Sepharose resulted in separation of the cytosolic fraction into two fractions, the flow-through and bound fractions, which synergistically supported the O2- production with the membrane fraction in the absence of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), whereas only the bound fraction besides the membrane fraction was required for the activation in the presence of GTP gamma S. The effective factors in the bound fraction were further purified by gel filtration on Superdex G-200 and anion exchange chromatography on Mono Q and found to be p47-phox and p63-phox. The purified recombinant p47-phox and p65-phox replaced corresponding native factors for the activation. These results suggest that the membrane fraction from pig neutrophils contains the GTP-binding protein responsible for the activation. Furthermore, the presence of the GTP-binding protein for the activation in the flow-through fraction from 2',5'-ADP-Sepharose was also shown on the basis of the findings that extensive dialysis of the flow-through fraction resulted in complete loss of the ability to activate the oxidase with the recombinant factors and the washed membrane of human neutrophils which contained no GTP-binding protein for the activation and the lost ability was recovered by the addition of GTP gamma S. Thus, activation of the oxidase in the cell-free system of pig neutrophils absolutely requires the GTP-binding protein which localizes in the membrane fraction or in the cytosolic fraction.

Animals↗
PubMed1993

Cyclic AMP inhibits the respiratory burst of electropermeabilized human neutrophils at a downstream site of protein kinase C.

We studied a signaling pathway for the activation of the superoxide (O2-)-generating NADPH oxidase and effects of cAMP on the pathway using electropermeabilized human neutrophils. The permeabilized cells produced O2- by the addition of protein kinase C (PKC) activator, phorbol myristate acetate (PMA), and a non-hydrolyzable GTP analogue, GTP gamma S in the presence of ATP and Mg2+. The O2- production by PMA not by GTP gamma S was inhibited by inhibitors of PKC. The production by PMA and GTP gamma S was inhibited by a GDP analogue, GDP beta S, in the same dose-dependent manner and the production by PMA was not enhanced by the addition of GTP gamma S and vice versa. These findings suggest the presence of a GTP-binding protein which follows PKC in the activation pathway. The O2- production by PMA and GTP gamma S was dose-dependently inhibited by cAMP and the inhibition was completely restored by an inhibitor of cAMP-dependent protein kinase, H-89, indicating that cAMP blocks the activating pathway at the site between the GTP-binding protein located downstream of PKC and the NADPH oxidase by activating cAMP-dependent protein kinase. The activation of the oxidase by sodium dodecyl sulfate (SDS) seemed to be different from the above pathway. It needed higher concentrations of GDP beta S for inhibition, did not absolutely need ATP and was inhibited by neither cAMP nor protein kinase C inhibitors. Moreover, the O2- production by the combination of GTP gamma S and SDS or of PMA and SDS was essentially the same as the sum of the production by each stimulant alone. We may conclude from the observations that the signaling pathway involving PKC for the activation of the oxidase is distinct from the pathway induced by SDS: the former is blocked by cAMP at the site between the GTP-binding protein located downstream of PKC and the oxidase and the latter is cAMP-insensitive.

Binding Sites↗
PubMed1993

Tyrosine phosphorylation is involved in the respiratory burst of electropermeabilized human neutrophils at a step before diacylglycerol formation by phospholipase C.

We studied a step where tyrosine phosphorylation is involved in a signaling pathway for the activation of the superoxide (O2-)-generating NADPH oxidase using electropermeabilized human neutrophils. The permeabilized cells produced O2- by the addition of a protein tyrosine phosphatase inhibitor, vanadate, as well as N-formyl-methionyl-leucyl-phenylalanine (fMLP) and protein kinase C (PKC) activators such as phorbol myristate acetate (PMA) and L-alpha-1-oleoyl-2-acetoyl-sn-3-glycerol (OAG). The O2- production by the stimulants was completely inhibited by PKC inhibitors such as calphostin C and staurosporine and was not affected by 1% ethanol, a metabolic modulator of phospholipase D (PLD). Furthermore, the O2- production by vanadate and fMLP, but not by OAG and PMA, was inhibited by both an inhibitor of phospholipase C (PLC), neomycin, and an inhibitor of tyrosine kinase, ST-638. These findings suggest that tyrosine phosphorylation is involved in the activation of the oxidase at a step before diacylglycerol formation by PLC, and that PLD may not be involved in the signaling pathway in permeabilized cells.

Cell Membrane Permeability↗
PubMed1993

Characterization of NAD(P)H-dependent ubiquinone reductase activities in rat liver microsomes.

Exogenous ubiquinone-10 was efficiently reduced by rat liver microsomes in the presence of NADH and NADPH under anaerobic conditions. Ubiquinone-10 reduced under anaerobic conditions was rapidly re-oxidized by the re-aeration. The reduction and re-oxidation were not observed when the reactions were carried out with the boiled microsomes or without microsomes, suggesting that the reactions were enzymatically catalyzed by the electron transport system(s) from NAD(P)H to O2 through the ubiquinone. The Km and Vmax of the reductase activity for NADH were 0.4 mM and 1.7 nmol/min per mg of protein, and those for NADPH were 19 microM and 2.1 nmol/min per mg of protein, respectively. The NADH-dependent oxidoreduction system was different from the NADPH-dependent system because of the following observations; (1) rotenone inhibited only the NADH-dependent ubiquinone-10 reductase, (2) dicoumarol inhibited the NADPH-dependent ubiquinone-10 reduction more potently than the NADH-dependent reduction and (3) the activity oxidizing the reduced ubiquinone-10 in the presence of NADH was less than that in the presence of NADPH. Endogenous ubiquinone-9 was also reduced and re-oxidized in essentially the same manner as exogenous ubiquinone-10. Thus, ubiquinone-10 oxidoreductase in rat liver microsomes acts on endogenous ubiquinone-9.

Animals↗
PubMed1993

The trans-sphincteric and trans-sacral approaches for the surgical excision of rectal and presacral lesions.

In the decade from 1981 to 1990, 30 patients underwent a posterior transsacral approach at the Aichi Medical University Hospital for their benign or malignant rectal lesions. The operation was classified into two procedures, consisting of the transsphincteric approach and transsacral approach, in order to cope with the condition of the anal sphincter muscles; whether they were divided or not. Eleven rectal tumors were successfully excised through the opened-up rectum by using the transsphincteric approach, and excellent results were obtained without any postoperative complications. Using the transsacral approach, 2 presacral dermoid cysts and 11 rectal lesions were easily removed under direct vision. Their prognoses were excellent. The transsacral approach was also applied for the resection of recurrent rectal cancers after a radical, abdominoperineal resection in 6 patients suffering from intolerable local symptoms. All the patients were free from these uncomfortable local symptoms after the surgery. The posterior transsacral operation is thus considered to be of value not only for resecting benign rectal and presacral lesions, but also for resecting malignant rectal tumors in frail subjects who are unfit for radical operation and/or recurrent rectal cancer.

Adenoma, Villous↗
PubMed1993

Conversion of xanthine dehydrogenase to xanthine oxidase during ischemia of the rat small intestine and the effect of trifluoperazine on the conversion.

The conversion from xanthine dehydrogenase (XD) to xanthine oxidase (XO) and the effect of trifluoperazine (TFP), a calmodulin inhibitor, on the conversion were examined during the normothermic ischemia of the rat small intestine. Rat jejunums were stored in lactated Ringer's solution (LR) at 37 degrees C for various hours after intravascular flushing with LR. The extents of the conversion from XD to XO (%XO) constituted 21.1% +/- 3.0%, 36.2% +/- 7.0%, 63.2% +/- 8.1%, and 88.2% +/- 8.6% after 0, 2, 4, and 6 hours of the preservation, respectively (control group). The preservation without the intravascular flushing showed significant increase in the %XO (99.5% +/- 6.0%) only after 6 hours compared with those in the control group (P < .05). When the intestines were stored in LR containing 50 mg/L of TFP at 37 degrees C, or stored in LR at 37 degrees C after the intraperitoneal pretreatment with 10 mg/kg of TFP 1 hour before laparotomy showed significant decrease in the extents of the conversion after 4 hours (P < .005) and 6 hours (P < .025) of the preservation, compared with those in the control group. When the dose of TFP for the pretreatment was increased to 50 mg/kg, the suppressive effect on the conversion was found even after 2 hours (P < .025) as well as after 4 hours (P < .005) and 6 hours (P < .025) of the preservation. These results suggest that TFP could be effective on reducing the XO-mediated postischemic reperfusion injury by means of inhibiting the conversion during ischemia of the rat small intestine.

Animals↗
PubMed1993

[Efficacy of 48-hour infusion of 5-fluorouracil for gall bladder cancer].

Usefulness of an adjuvant chemotherapy for 23 patients who had undergone "non-curative" resection for adenocarcinomas of the gallbladder was investigated. Patients were divided into two groups: Group A (16 patients) was given MF (mitomycin C 6 mg/m2, 5-fluorouracil 250 mg/body) by injection on the 2nd and 9th postoperative day and received orally Tegafur 600 mg/day and PSK 3 g/day. Group B(7 patients) was treated weekly with a 48-hour infusion of 5-FU (1,000 mg/m2/24 hours), for 6 weeks and leucovorin (30 mg/body) was given for 2 hours prior to 5-FU infusion. The results were evaluated by the Kaplan-Meier method. Response rate of anti-tumor was 0% in Group A and 14.3% in Group B, including: PR, 1 and NC, 6 cases. The 50% survival time was 230 days in group A and 471 days in group B (p = 0.0008). The results suggest that treatment with 5-FU and LV is effective for gallbladder cancer.

Adenocarcinoma↗
PubMed1992

Cytochrome b558, a component of the phagocyte NADPH oxidase, is a flavoprotein.

Cytochrome b558 is the only membrane component of the phagocyte O2(-)-producing NADPH oxidase. The O2- production by the oxidase reconstituted in vitro with the crude membrane fraction is enhanced several-fold by addition of FAD, whereas that with the partially purified cytochrome is completely dependent on exogenous FAD, suggesting that FAD acts through the membrane component, cytochrome b558. The alignments of the amino acid sequence of the large subunit of the cytochrome (gp91-phox) with those of previously characterized flavoproteins reveal that the middle and C-terminal portions of gp91-phox are likely to be FAD- and NADPH-binding domains, respectively. Cytochrome b558, thus, appears to be a flavoprotein with an NADPH-binding site, of the NADPH oxidase.

Amino Acid Sequence↗
PubMed1992

Role of Mg2+ in activation of NADPH oxidase of human neutrophils: evidence that Mg2+ acts through G-protein.

The membrane fraction and three cytosolic proteins of neutrophils, p47-phox, p67-phox and a G-protein, are involved in the cell-free activation of the O2(-)-generating NADPH oxidase in the presence of SDS, though it has been controversial whether the G-protein is required or just enhancing the activity. We have used the three cytosolic factors, the solubilized membrane fraction, GTP gamma S and SDS, and found that both G-protein and GTP gamma S are essential for the activation of the NADPH oxidase. The effect of GTP gamma S is modified by Mg2+: the cations enhance the O2- generation at low concentrations of GTP gamma S, whereas they attenuate the activity at higher concentrations of GTP gamma S. In presence of 10 microM GTP gamma S, the maximal activity is observed at 0.1 microM Mg2+, which is several-fold higher than that at 1 mM Mg2+. The omission of Mg2+ followed by the chelation with EDTA results in loss of the activation, which is completely restored by the addition of Mg2+. Thus, Mg2+ seems to modulate the activation of the NADPH oxidase at the level of the G-protein.

Antibodies, Monoclonal↗